CN108761093A - A kind of antibodies against foot-and-mouth disease virus evaluation test strips - Google Patents
A kind of antibodies against foot-and-mouth disease virus evaluation test strips Download PDFInfo
- Publication number
- CN108761093A CN108761093A CN201810796432.6A CN201810796432A CN108761093A CN 108761093 A CN108761093 A CN 108761093A CN 201810796432 A CN201810796432 A CN 201810796432A CN 108761093 A CN108761093 A CN 108761093A
- Authority
- CN
- China
- Prior art keywords
- mouth disease
- disease virus
- foot
- test strips
- gold
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of antibodies against foot-and-mouth disease virus to evaluate test strips, the detection line of the test strips is printed using FMDV VP1 epitope polypeptide artificial antigen, and selected polypeptide is FMDV VP1 epitope polypeptide (the 142nd~158 amino acids sequence).Artificial antigen prepared by the present invention, which has, to be easy to get, stable structure, purity is at low cost up to 99%, can rapid, high volume production the advantages that, it is low that the use of this artificial antigen solves tradition expression expressing quantity, it is difficult to ensure that natural space structure, renaturation are difficult, it is difficult to remove mycoprotein, the shortcomings of influencing the specificity of testing result, also solve that there are the infull risks of inactivation of virus using inactivation of viruses.The present invention also pre-processes gold-labelled pad, is more advantageous to gold-labelled pad water suction and the release of gold mark albumen, and it is slow and not exclusively effectively to solve existing for existing test paper gold mark protein delivery, the problems such as influencing test paper product testing accuracy, sensitivity and shelf-life.
Description
Technical field
The present invention relates to a kind of antibodies against foot-and-mouth disease virus to evaluate test strips, belongs to technical field of immunoassay.
Background technology
Aftosa is to cause a kind of strong infectiousness epidemic disease disease artiodactylous by foot and mouth disease virus.Foot and mouth disease virus has
The extremely strong property of polymorphism, mutability, host's popularity, contagiousness, so once morbidity is i.e. in popular, great outburst.It is more tight
Weight, aftosa is a kind of deadly infectious disease, according to there is extremely strong infectiousness, for the same group of affected animal and contact cause of disease
Animal, it is necessary to be strictly isolated, be blocked, forbid animal movement and livestock products listing etc., therefore, Infected regions is caused even to be sent out
The livestock products foreign trade of sick country stops, and causes huge economic loss.
The main policies of developing country's control aftosa are vaccine immunities, and it is dynamic that animal population vaccine immunity is immunized in aftosa
The formulation of object immune effect, maternal antibody and immune programme is required for relying on antibody test.Antibodies against foot-and-mouth disease virus water at present
The serological method of flat detection is mainly Liquid-phase blocking ELISA (Liquid phase blocking ELISA, LPBE) and resists
Body test strip.Antigen used in these methods is mainly inactivation of viruses or expression albumen, and there are viruses to go out for inactivation of viruses
The problem of incomplete, purification difficult living；Expression albumen there are this expressing quantities the problems such as low, mycoprotein is difficult to remove.Antibody
Test strip has quick, easy, low-cost advantage, and gold-labelled pad is the important component of test paper, it determines gold
The activity and release property of albumen are marked, therefore very big to the influential effect of colloidal gold immune chromatography test.Currently, the preparation of gold-labelled pad
Method is albumen/antibody of direct spraying or immersion colloid gold label on cellucotton, or cellucotton is impregnated using treatment fluid
After processing, then spray or impregnate the albumen of colloid gold label.But it finds in use, because of gold-labelled pad processing method and processing
Formula of liquid is improper, often exist gold mark protein delivery it is slow and not exclusively, to influence test paper product testing accuracy, sensitivity
And the problems such as shelf-life.
Invention content
In order to overcome in the prior art expression expressing quantity it is low, it is difficult to ensure natural space structure, renaturation is difficult, bacterium
Body protein influences specificity and inactivation of viruses the shortcomings of there are inactivation of virus infull risks and the existing examination of testing result
Gold mark protein delivery existing for paper is slow and not exclusively, the problems such as influencing test paper product testing accuracy, sensitivity and shelf-life, this
Invention combine existing aftosa detection ELISA method and test strips advantage and disadvantage, using polypeptide epitope prepare artificial antigen and
Gold-labelled pad processing method, a kind of novel antibodies against foot-and-mouth disease virus of invention evaluate test strips.
To achieve the goals above, the technical solution adopted in the present invention is：
The detection line of a kind of antibodies against foot-and-mouth disease virus evaluation test strips, test strips is more using FMDV VP1 epitope
Peptide artificial antigen is printed, and VP1 epitope polypeptide artificial antigens are artificial synthesized FMDV VP1 epitope polypeptide, and are coupled
The artificial antigen that carrier protein is formed.
FMDV VP1 epitope polypeptide be FMDV VP1 on the 142nd~158 amino acids sequence, and its-
NH4A cysteine is added in end.
Foot and mouth disease virus is foot and mouth disease virus O/GX/09-7 strains, foot and mouth disease virus O/HN/CHA/93 strains, aftosa
At least one of virus O/TAW/97 strains, foot and mouth disease virus O/MYA/98 strains.
Upper 142nd~158 amino acids sequences of foot and mouth disease virus O/GX/09-7 strains VP1 are
NNVRGDLQVLAQKAERA；Upper 142nd~158 amino acids sequences of foot and mouth disease virus O/HN/CHA/93 strains VP1 are
SNVRGDLQVLAQKAERA；Upper 142nd~158 amino acids sequences of foot and mouth disease virus O/TAW/97 strains VP1 are
NNVRGDLQVLAQKAERT；Upper 142nd~158 amino acids sequences of foot and mouth disease virus O/MYA/98 strains VP1 are
TNVRGDLQVLAQKAARP。
Antibodies against foot-and-mouth disease virus evaluates test strips, further includes the control using the two anti-igg printing for resisting animal species to be checked
Line.
Test strips include sample pad, gold-labelled pad, absorption pad and bottom plate；Detection line and control line are printed on nitrocellulose filter
Pad.
Gold-labelled pad is pre-processed, treatment fluid is sprayed on fiber mat, it is dry, obtain pretreated gold-labelled pad.
Treatment fluid is made of following raw material：BSA 5%, PVP-10 0.1%, Triton X-100 0.1%, surplus are
The Na of 0.02mol/L2B4O7·10H2O solution.
Spraying method is：Along the center spray treatment liquid of fiber mat length direction, and the treatment fluid after spraying on fiber mat
There is gap between in the side of both sides of the edge line and fiber mat respective side.
Dry condition is：37-42 DEG C, 10-70min.
Advantageous effect of the present invention：
1, detection line antigen used in test paper of the present invention uses the epitope polypeptide of synthesis, artificial anti-after coupling carrier albumen
Original has and is easy to get, and stable structure, purity is at low cost up to 99%, can rapid, high volume production the advantages that, it is this artificial anti-
It is low that former use solves tradition expression expressing quantity, it is difficult to ensure that natural space structure, renaturation are difficult, it is difficult to remove bacterium
Body protein, also solves that there are the infull risks of inactivation of virus using inactivation of viruses at the shortcomings of influencing the specificity of testing result.
2, in the test paper pattern of multi-joint antibody test, gold mark albumen can only select can be in conjunction with the albumen of mammal IgG
(such as SPA) or antiantibody.In the delivery system of the present invention, control line is two anti-igg for resisting animal species to be detected, secondary antibody
IgG can ensure that control line develops the color in conjunction with gold mark thing and in conjunction with the IgG- gold mark things that not tested survey line antigen protein intercepts
Stability.
3, be formulated in gold-labelled pad treatment fluid of the present invention it is simple, without sugar, be more advantageous to gold-labelled pad water suction and gold mark albumen
Release.Wherein, BSA and PVP-10 can close cellucotton and NC films, while protect the stability of gold mark albumen；Na2B4O7·
10H2O can provide alkali ion environment, more conducively antigen-antibody reaction and the release of gold mark albumen；Triton X-100 are used for
The processing of gold-labelled pad can accelerate the release of gold mark albumen, and can reduce the non-specificity of product.Treatment fluid selection of the present invention
The reason of not sugaring：Sugar is easy to crystallize during product storage, can influence the golden release for marking albumen and gold particle in film
On movement speed, to influence the shelf life and detection accuracy of product.
4, method disclosed by the invention is different from the existing method for impregnating gold-labelled pad in treatment fluid, but direct spraying
On glass fibre cotton, the mode of spraying is selected to substitute dipping pretreatment gold-labelled pad, can be effectively improved and impregnate gold-labelled pad generation
Edge effect；The mode of spraying can make treatment fluid in local fixation, and gold-labelled pad rest part can be allowed to keep fluffy state, increase
Add the water imbibition and adhesiveness of gold-labelled pad, applicability more extensive；The mode of spraying is fixed on one after can making gold mark albumen spraying
Determine to spread without large area in range, is more advantageous to stabilization and the release of gold mark albumen, greatly improves colloidal gold immunochromatographimethod examination
The detection sensitivity and stability of paper.
5, easy to operate, quick：Using ELISA test strip of the present invention in the process without adding any other instrument and reagent,
As long as its test lead is inserted into 30s or so in serum to be checked, then testing result is can determine that in 5min or so.
6, it reduces investment and reduces testing cost：Using Rapid detection test strip of the present invention, need not separately match Other Instruments,
Equipment and reagent save big measuring appratus, equipment and additive reagent expense；One test paper once can both detect aftosa and group is immunized
The antibody level of body, and infection of foot-and-mouth disease can be detected or with malicious animal, and also profession and layman can be whenever and wherever possible
Real-time online detection is carried out, without paying expert diagnosis Laboratory Fee and its correlative charges.Therefore, testing cost can be saved, is dropped
Low testing cost.
7, have a wide range of application, convenient for promoting the use of：Rapid detection test strip of the present invention is easy to operate at " single step " or " stupid
Melon formula ", and be convenient for carrying and preserve, the needs of different levels personnel, including professional chemical examination, customs quarantine control, health can be met
Epidemic prevention, quality-monitoring, livestock products processing, intensive culture to individual cultivation etc., have a vast market foreground and larger warp
Ji, social benefit.
Description of the drawings
The specific implementation mode of the present invention is described in further detail below in conjunction with attached drawing.
Fig. 1：Antibodies against foot-and-mouth disease virus evaluates test strips specific assay result.
Fig. 2：Antibodies against foot-and-mouth disease virus evaluates test strips sensitivity test result.
Fig. 3：The effect of 1 processed gold-labelled pad of embodiment and not pretreated gold-labelled pad spraying gold mark albumen.
Fig. 4：The gold-labelled pad situation of control methods processing.
Fig. 5：The service condition of embodiment 2, the gold-labelled pad of control methods processing.
Specific implementation mode
The specific implementation mode of the present invention is described in further detail with reference to embodiments.Unless otherwise specified, originally
" % " in invention specific implementation mode is quality percent by volume (g/mL).
Embodiment 1
Prepare antibodies against foot-and-mouth disease virus evaluation test strips：It is artificial synthesized first according to B cell epitope on FMDV VP1
Epitope polypeptide on Foot-and-mouth disease VP1, and coupling carrier albumen, while preparing the secondary antibody for resisting animal species to be checked
IgG, the detection line (T) for being respectively used to prepare on cellulose membrane pad and control line (C)；It prepares gold-labelled pad treatment fluid and gold-labelled pad is pre-
Processing, for spraying gold mark albumen；Finally assemble test strips.
1, the synthesis of Foot-and-mouth disease epitope polypeptide：
Synthesizing the 142nd~158 amino acids sequence on FMDV VP1, (amino acid sequence is according to examined aftosa poison
Strain is different and different), in its-NH4A cysteine is added in end, and polypeptide sequence is synthesized and purified by commercial company.
2, the preparation of Foot-and-mouth disease epitope polypeptide artificial antigen：
With Heterobifunctional reagents Sulfo-SMCC (MW：436.37 Spacer Arm Length：Pierce)
- NH on activated carrier protein B SA (or OVA)2, concrete operation step is as follows：
(1) coupling buffer (50mL)：0.15M NaCl, 0.1M PB buffer solutions (pH 7.2), 1 μM of EDTA (ethylenediamine
Tetraacethyl)；
(2) BSA (bovine serum albumin(BSA)) solution：The carrier protein BSA for weighing 8mg is dissolved in 1.0mL coupling buffers；
(3) Sulfo-SMCC solution：It weighs 2mg Sulfo-SMCC to be added in the DMSO (dimethyl sulfoxide (DMSO)) of 100 μ L, instead
Multiple piping and druming, makes it fully dissolve；
(4) BSA solution and Sulfo-SMCC solution are mixed, after mixing well, at room temperature, react 1h or 37 DEG C,
30min, and mixing frequently；
(5) dialysis 48h is carried out to the solution of step (4) under the conditions of 4 DEG C with coupling buffer, changes the liquid once, goes per 6h
Except extra coupling agent (Sulfo-SMCC) and DMSO；
(6) carrier protein BSA concentration is adjusted to 5mg/mL with coupling buffer, which is SMCC activated carrier albumen
(SMCC-BSA), -20 DEG C freeze it is spare.
- NH on carrier protein2It is connected with-the SH of polypeptide (Pep) N-terminal cysteine (Cys) after activated, forms people
Work combination antigen (BSA-Pep).Coupling step is：4mg polypeptides are weighed, after fully being dissolved with the PBS buffer solution of 0.01M (for
Dissolubility relatively low polypeptide DMF or DMSO dissolves)；300 μ L 0.01M PB buffer solutions are added, and (pH 7.2 contains 5mM
EDTA), polypeptide storing liquid, a concentration of 10mg/mL are prepared.When coupling, 20 μ L polypeptide storing liquids are taken, is added and contains 5mM in equal volume
In the 0.01M PB buffer solutions (pH 7.2) of EDTA, adds 40 μ L SMCC-BSA solution and be sufficiently mixed, after reacting at room temperature 4h, 4
DEG C be incubated overnight.Dialysis 48h is carried out to above-mentioned solution under the conditions of 4 DEG C with physiological saline, is changed the liquid once per 6h, it is extra to remove
Coupling agent；Its albumen concentration is measured with ultraviolet specrophotometer.
3, goat-anti or rabbit-anti are detected the preparation of the secondary antibody of animal species IgG：
With the 50 μ g IgG/kg weight of μ g~100 through subcutaneous and intramuscular injection negative healthy sheep or rabbit 3~4 times, last is exempted from
Venous blood collection after epidemic disease 20 days measures its serum antibody titer 1 with ELISA：2000 or more, Culling heart blood or arteria carotis bloodletting,
Collect its hyper-immune serum.It takes 1 volume of serum to add 2 volume PBS buffer solution (pH 7.2) mixings, adds isometric saturated ammonium sulfate liquid mixed
It is even, 2h in 4 DEG C of refrigerators is set, 15min is centrifuged in 4 DEG C, 10000r/min, abandons supernatant；It is molten with appropriate PBS buffer solution (pH7.2)
Solution precipitation, it is 33% to add saturated ammonium sulfate liquid to its ultimate density, 2h in 4 DEG C of refrigerators is set, under the conditions of 4 DEG C, 10000r/min
15min is centrifuged, supernatant is abandoned, is dissolved and is precipitated with a small amount of PBS buffer solution (pH7.2), set in 4 DEG C of refrigerators and use PBS buffer solution
(pH7.2) it is dialyzed overnight, changes liquid 2~3 times, 15min is centrifuged under the conditions of 4 DEG C, 10000r/min, supernatant is collected, with ultraviolet
Its albumen concentration of spectrophotometric determination.
4, the preparation of gold mark albumen：
Aurosol is prepared with reduction of sodium citrate method：I.e. in 0.01~0.05% chlorine of mass fraction of 50~100mL boilings
2~4mL mass fractions, 0.5~2% citric acid three sodium solution is added in auric acid aqueous solution, obtains the colloid of diameter 15nm or so
Gold.With the K of 0.1mol/L2CO3Colloidal gold pH to 8.5~9.5 is adjusted, with 1：1000~1300 label is than by albumen to be marked
SPA is added in the colloidal gold of pH8.5~9.5, after marking 10min, adds the mass fraction 20%PEG-10000 to be to its ultimate density
0.05%, 4 DEG C, 1500~3000r/min centrifugation 20min remove unbonded colloid gold particle, 4 DEG C, 15000r/min centrifugations
1h abandons supernatant, obtains colloid gold label albumen.
5, the preparation of gold-labelled pad：
Gold-labelled pad preprocess method：Treatment fluid is sprayed on the amount of 8 μ L/cm on fiber mat using spraying apparatus, 42 DEG C
50min is dried, pretreated gold-labelled pad is obtained.Gold-labelled pad treatment fluid is made of following raw material：BSA5%, PVP-10 0.1%,
Triton X-100 0.1%, surplus are the Na of 0.02mol/L2B4O7·10H2O solution.Spraying method is：Along fiber mat length
The center spray treatment liquid in direction, and the side of the treatment fluid both sides of the edge line and fiber mat respective side after spraying on fiber mat is along it
Between there is gap.Then the colloid gold label albumen of preparation is uniformly sprayed in gold-labelled pad, 42 DEG C of baking 40min.
6, the assembling of test strips
Foot-and-mouth disease epitope polypeptide artificial antigen and goat-anti or rabbit-anti are detected the two of animal species IgG
It is anti-to be sprayed on nitrocellulose filter respectively, form detection trace/line (T) and control trace/line (C)；By sample pad, Jin Biao
Pad, cellulose membrane pad, absorption pad are pasted on bottom plate successively, are cut into suitable dimension and are obtained test paper.
It is printed with red samples mark line on sample pad face layer corresponding with gold-labelled pad intersection, and is printed on max printed words,
The label alerts at the side 1.1-1.2cm of linear distance sample pad top.
The principle of Rapid detection test strip examinations of the present invention：
After Rapid detection test strip test lead of the present invention is inserted into serum to be detected, serum to be checked is driven to be checked by siphon
Gold mark albumen in serum antibody and gold mark albumen mineral wool eventually penetrates handle end filter together to nitrocellulose membrane diffusion
In paper, antibody can be combined with gold mark albumen in diffusion process, which detects with the artificial antigen on cellulose membrane in turn
Trace combines, to show the detection trace (T) of brownish red；And control line trace can be combined with gold mark thing, form brownish red
Compare trace (C).If not having antibodies against foot-and-mouth disease virus and/or vaccine antibody in serum to be checked, test strips only show one
(a) brownish red compares trace (C)；If in serum to be checked contain Anti-FMDV antibody and/or vaccine antibody, first with
Its gold mark protein binding, then combined with the artificial antigen detection trace (T) on cellulose membrane, show the detection print of brownish red
Mark is positive mark；If there is no any brownish red trace to show on cellulose membrane, show that test strips have failed or operated mistake
Accidentally.
Embodiment 2
Antibodies against foot-and-mouth disease virus evaluates the preparation method such as embodiment 1 of test strips, and foot and mouth disease virus O/ is sprayed in detection line
Upper 142nd~158 amino acids sequence NNVRGDLQVLAQKAERA of GX/09-7 strains VP1 (SEQ ID NO.1), in its-NH4
A cysteine, artificial antigen prepared by coupling carrier protein B SA or OVA is added in end.
The detection operating method of test strips：
(1) preparation of sample liquid is detected：It is sterile to take the blood of tested animal, and serum is detached, make 1 with physiological saline：200
Times dilute serum is to be measured；Rapid detection test strip test lead of the present invention is inserted into serum to be detected, insertion depth is no more than mark
Remember line, test strips are taken out after about 30s, is horizontally arranged about 1~5min, while observing result.
(2) result judges：If only showing (a) brownish red control print in test strip cellulose film layer
Mark (C) indicates that testing result is negative, and illustrate not detect antibodies against foot-and-mouth disease virus in tested serum, that is, be detected animal without
Mouth disease virus infection and/or aftosa vaccine are immune；If there is brownish red in the cellulose film layer in test strip
Trace (C) and detection trace (T) are compareed, indicates that testing result is positive, i.e., detects that foot and mouth disease virus is anti-in serum to be checked
Body, i.e., tested animal have carried out aftosa vaccine and have been immunized or have infected foot and mouth disease virus；If not any on cellulose membrane band
Brownish red trace is shown, then shows that test strips have failed or operated wrong.
Embodiment 3
Preparation method such as embodiment 2, the difference that antibodies against foot-and-mouth disease virus evaluates test strips are manually to resist in detection line
Former polypeptide is upper 142nd~158 amino acids sequence SNVRGDLQVLAQKAERA of foot and mouth disease virus O/HN/CHA/93 strains VP1
(SEQ ID NO.2)。
Embodiment 4
Preparation method such as embodiment 2, the difference that antibodies against foot-and-mouth disease virus evaluates test strips are manually to resist in detection line
Former polypeptide is upper 142nd~158 amino acids sequence NNVRGDLQVLAQKAERT of foot and mouth disease virus O/TAW/97 strains VP1
(SEQ ID NO.3)。
Embodiment 5
Preparation method such as embodiment 2, the difference that antibodies against foot-and-mouth disease virus evaluates test strips are manually to resist in detection line
Former polypeptide is upper 142nd~158 amino acids sequence TNVRGDLQVLAQKAARP of foot and mouth disease virus O/MYA/98 strains VP1
(SEQ ID NO.4)。
Embodiment 6
Preparation method such as embodiment 2, the difference that antibodies against foot-and-mouth disease virus evaluates test strips are manually to resist in detection line
Former polypeptide selects upper 142nd~158 amino acids sequences of foot and mouth disease virus O/GX/09-7 strains VP1 simultaneously
NNVRGDLQVLAQKAERA, upper 142nd~158 amino acids sequences of foot and mouth disease virus O/HN/CHA/93 strains VP1
SNVRGDLQVLAQKAERA, upper 142nd~158 amino acids sequences of foot and mouth disease virus O/TAW/97 strains VP1
NNVRGDLQVLAQKAERT and upper 142nd~158 amino acids sequences of foot and mouth disease virus O/MYA/98 strains VP1
TNVRGDLQVLAQKAARP, four kinds of polypeptides in mass ratio 1:1:1:1 mixing.
Verify example
1, specific detection
With the test paper detection aftosa positive serum, aftosa negative serum and swine fever virus, pig blue-ear disease of the present invention
Virus, pig circular ring virus, porcine pseudorabies virus, Latex agglutination test positive serum, the results show that test paper of the present invention is special
Property is 100%, with aftosa negative serum and other virus-positive serum no cross reactions (such as Fig. 1).
2, sensitivity technique
The standard Schweineseuche immune serum that doubling dilution is detected with the test paper of the present invention, as a result shows test strips of the present invention
Sensitivity be 1:12800 (such as Fig. 2).
Contrast experiment
1, gold mark albumen (spraying position and treatment fluid spraying position phase are sprayed in 1 processed gold-labelled pad of embodiment
Together), gold mark albumen is fixed at a line of aggregation in gold-labelled pad；And gold mark albumen is sprayed in not pretreated gold-labelled pad
(spraying position is identical as processed gold-labelled pad position), gold mark albumen are saturated with entire gold-labelled pad at disperse shape, illustrate the present invention
The gold-labelled pad of processing can allow the proteopexy of gold mark in a certain range, be more advantageous to stabilization and the release (Fig. 3) of gold mark albumen.
2, as a comparison with the preprocess method of other gold-labelled pads, compared with the pretreating effect of gold-labelled pad of the present invention
Compared with.The preprocess method of comparison gold-labelled pad is that fiber mat is immersed in 15min in treatment fluid, then takes out and is dried at 37 DEG C, is obtained
To pretreated gold-labelled pad.Treatment fluid is made of following raw material：BSA 3%, trehalose 8%, Tween-20 2%, surplus are
0.01mol/L phosphate buffers.As a result it known to (Fig. 4), is unevenly distributed after the gold-labelled pad drying of control methods processing, edge
There is processed material precipitation jaundice, and quality is harder, can not adhere on the supporting plate, cut because containing Tween-20 in treatment fluid
When be easy to happen gold-labelled pad obscission, this disadvantage can be improved using the method for spray treatment.
3, the SPA of colloid gold label is sprayed in embodiment 2 and the pretreated gold-labelled pad of control methods, and test paper inspection is made
Infection of foot-and-mouth disease serum is surveyed, is observed after 10min.Gold mark protein delivery rate is 95% or more in 2 gold-labelled pad of embodiment, control methods
Gold-labelled pad on gold mark SPA do not discharge completely, and test strips colour developing be weaker than embodiment 2 (Fig. 5).
The foregoing is merely the embodiments that the present invention is best, and for those skilled in the art, the present invention can have
Various modifications and variations.All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on, should all
It is included within protection scope of the present invention.
Sequence table
<110>Henan Bao'ao Biology Engineering Co., Ltd
<120>A kind of antibodies against foot-and-mouth disease virus evaluation test strips
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 17
<212> PRT
<213>Foot and mouth disease virus (Footand Mouth Disease Virus)
<400> 1
Asn Asn Val Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Ala Glu Arg
1 5 10 15
Ala
<210> 2
<211> 17
<212> PRT
<213>Foot and mouth disease virus (Footand Mouth Disease Virus)
<400> 2
Ser Asn Val Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Ala Glu Arg
1 5 10 15
Ala
<210> 3
<211> 17
<212> PRT
<213>Foot and mouth disease virus (Footand Mouth Disease Virus)
<400> 3
Asn Asn Val Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Ala Glu Arg
1 5 10 15
Thr
<210> 4
<211> 17
<212> PRT
<213>Foot and mouth disease virus (Footand Mouth Disease Virus)
<400> 4
Thr Asn Val Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Ala Ala Arg
1 5 10 15
Pro
Claims (10)
1. a kind of antibodies against foot-and-mouth disease virus evaluates test strips, which is characterized in that the detection line of the test strips is to utilize aftosa
Virus VP 1 epitope polypeptide artificial antigen is printed, and VP1 epitope polypeptide artificial antigens are artificial synthesized FMDV VP1 table
Position polypeptide, and the artificial antigen that coupling carrier albumen is formed.
2. antibodies against foot-and-mouth disease virus according to claim 1 evaluates test strips, which is characterized in that FMDV VP1 table
Position polypeptide is the 142nd~158 amino acids sequence on FMDV VP1, and in its-NH4A cysteine is added in end.
3. antibodies against foot-and-mouth disease virus according to claim 2 evaluates test strips, which is characterized in that foot and mouth disease virus is mouth hoof
Epidemic disease poison O/GX/09-7 strains, foot and mouth disease virus O/HN/CHA/93 strains, foot and mouth disease virus O/TAW/97 strains, hoof-and-mouth disease
At least one of malicious O/MYA/98 strains.
4. antibodies against foot-and-mouth disease virus according to claim 3 evaluates test strips, which is characterized in that foot and mouth disease virus O/GX/
Upper 142nd~158 amino acids sequences of 09-7 strains VP1 are NNVRGDLQVLAQKAERA；Foot and mouth disease virus O/HN/CHA/93
Upper 142nd~158 amino acids sequences of strain VP1 are SNVRGDLQVLAQKAERA；Foot and mouth disease virus O/TAW/97 strains VP1
Upper 142nd~158 amino acids sequence is NNVRGDLQVLAQKAERT；Foot and mouth disease virus O/MYA/98 strains VP1 the upper 142nd
~158 amino acids sequences are TNVRGDLQVLAQKAARP.
5. antibodies against foot-and-mouth disease virus according to claim 1 evaluates test strips, which is characterized in that further include using anti-to be checked
The control line of the two anti-igg printing of animal species.
6. antibodies against foot-and-mouth disease virus according to claim 1 evaluates test strips, which is characterized in that the test strips further include
Sample pad, gold-labelled pad, absorption pad and bottom plate；Detection line and control line are printed on nitrocellulose filter pad.
7. antibodies against foot-and-mouth disease virus according to claim 6 evaluates test strips, which is characterized in that located in advance to gold-labelled pad
Reason, treatment fluid is sprayed on fiber mat, dry, obtains pretreated gold-labelled pad.
8. antibodies against foot-and-mouth disease virus according to claim 7 evaluates test strips, which is characterized in that treatment fluid is by following raw material
Composition：BSA5%, PVP-10 0.1%, Triton X-100 0.1%, surplus are the Na of 0.02mol/L2B4O7·10H2O is molten
Liquid.
9. antibodies against foot-and-mouth disease virus according to claim 7 evaluates test strips, which is characterized in that spraying method is：Along fine
The center spray treatment liquid of dimension pad length direction, and the treatment fluid both sides of the edge line after spraying on fiber mat and fiber mat respective side
Side along between there is gap.
10. antibodies against foot-and-mouth disease virus according to claim 7 evaluates test strips, which is characterized in that dry condition is：
37-42 DEG C, 10-70min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810796432.6A CN108761093B (en) | 2018-07-19 | 2018-07-19 | Test strip for evaluating foot-and-mouth disease virus antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810796432.6A CN108761093B (en) | 2018-07-19 | 2018-07-19 | Test strip for evaluating foot-and-mouth disease virus antibody |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108761093A true CN108761093A (en) | 2018-11-06 |
| CN108761093B CN108761093B (en) | 2020-12-25 |
Family
ID=63970471
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810796432.6A Active CN108761093B (en) | 2018-07-19 | 2018-07-19 | Test strip for evaluating foot-and-mouth disease virus antibody |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108761093B (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1991003255A1 (en) * | 1989-09-08 | 1991-03-21 | The Wellcome Foundation Limited | Polypeptide vaccines against foot-and-mouth disease virus |
| CN101724605A (en) * | 2008-10-30 | 2010-06-09 | 中国农业科学院哈尔滨兽医研究所 | Foot-and-mouth disease virus (FMDV) resistant monoclonal antibody and identified epitope and application thereof |
| CN104090107A (en) * | 2014-06-12 | 2014-10-08 | 广东省食品药品检验所 | Sulphonamide drug rapid screening method |
| CN104292300A (en) * | 2014-09-17 | 2015-01-21 | 复旦大学 | Epitope minimum motif peptide of P1, VP2 and VP4 structural proteins in type O foot and mouth disease virus (FMDV) strain (O/BY/CHA/2010) and application of epitope minimum motif peptide |
| CN104374909A (en) * | 2014-11-17 | 2015-02-25 | 北京工业大学 | Chloramphenicol quantitative detection method based on up-conversion phosphor technology and immunochromatography technology |
| CN105319363A (en) * | 2014-07-31 | 2016-02-10 | 加拿大莱博瑞医疗技术公司 | Mobile Phase Test Strip Components, Conjugate Pad Pre-treatment Solutions, And Related Methods Thereof |
-
2018
- 2018-07-19 CN CN201810796432.6A patent/CN108761093B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1991003255A1 (en) * | 1989-09-08 | 1991-03-21 | The Wellcome Foundation Limited | Polypeptide vaccines against foot-and-mouth disease virus |
| CN101724605A (en) * | 2008-10-30 | 2010-06-09 | 中国农业科学院哈尔滨兽医研究所 | Foot-and-mouth disease virus (FMDV) resistant monoclonal antibody and identified epitope and application thereof |
| CN104090107A (en) * | 2014-06-12 | 2014-10-08 | 广东省食品药品检验所 | Sulphonamide drug rapid screening method |
| CN105319363A (en) * | 2014-07-31 | 2016-02-10 | 加拿大莱博瑞医疗技术公司 | Mobile Phase Test Strip Components, Conjugate Pad Pre-treatment Solutions, And Related Methods Thereof |
| CN104292300A (en) * | 2014-09-17 | 2015-01-21 | 复旦大学 | Epitope minimum motif peptide of P1, VP2 and VP4 structural proteins in type O foot and mouth disease virus (FMDV) strain (O/BY/CHA/2010) and application of epitope minimum motif peptide |
| CN104374909A (en) * | 2014-11-17 | 2015-02-25 | 北京工业大学 | Chloramphenicol quantitative detection method based on up-conversion phosphor technology and immunochromatography technology |
Non-Patent Citations (1)
| Title |
|---|
| SUZHEN YANG等: "A rapid immunochromatographic strip for neutralizing antibodies detection of foot and mouth disease virus serotype O", 《RSC ADV》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108761093B (en) | 2020-12-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP2064551B1 (en) | Combination hepatitis c virus antigen and antibody detection method | |
| CN107255726B (en) | Quantitatively detect the fluorescence immune chromatography test paper and preparation method thereof of human parathyroid hormone | |
| CN102072957B (en) | Hepatitis C virus antibody diagnostic kit and preparation method thereof | |
| CN111233985A (en) | Preparation method of novel coronavirus IgA antibody rapid detection test strip | |
| CN108490166A (en) | A kind of improved experimental buffer solution and its application | |
| CN104198703A (en) | Human mycoplasma pneumoniae gold-marked silver-stained immunochromatographic assay kit and preparation method and application thereof | |
| CN101735319B (en) | Monoclonal antibody against GP73 protein, preparation method and application thereof | |
| CN109900902A (en) | A kind of porcine pseudorabies virus gB blocks ELISA antibody assay kit and its application | |
| CN101393216A (en) | Colloidal carbon test paper strip for rapid diagnosis of schistosomiasis japanica | |
| CN112946260B (en) | Fluorescence immunochromatography reagent for detecting novel coronavirus pneumonia virus antibody and preparation method thereof | |
| CN104090101A (en) | Human immunodeficiency virus (HIV) antibody detection kit and preparation method thereof | |
| CN101266248A (en) | HCV IgM antibody rapid detection test paper | |
| CN201413329Y (en) | AI9A antibody combined quick detecting test paper of Epstein-Barr virus capsid antigen VCA and thymidine kinase TK albumen immuneoglobrin | |
| CN106188248A (en) | A kind of Epstein-Barr virus antigen preparation procedure and utilize the quick detection kit of detection Epstein-Barr virus antibody prepared by this antigen | |
| CN108931644B (en) | Foot-and-mouth disease virus immune antibody evaluation and infection and immune differential diagnosis dual test strip | |
| CN109212239A (en) | A kind of giant panda luteotropin colloidal gold immuno-chromatography test paper strip, preparation method and application | |
| CN108761093A (en) | A kind of antibodies against foot-and-mouth disease virus evaluation test strips | |
| CN106932592A (en) | Detect colloidal gold strip of people's surfactant protein A and its preparation method and application | |
| CN108982845B (en) | A differential diagnosis test strip between a foot-and-mouth disease virus-infected animal and a vaccine-immunized animal | |
| CN107402300A (en) | A kind of method of quick discriminating human parathyroid | |
| CN101509002B (en) | Recombinant rubella virus protein and uses thereof | |
| CN109633162A (en) | S-100 β protein detection kit | |
| CN1632585A (en) | Immune chromatographic test paper for detecting brucellar infection and preparing process thereof | |
| CN110133285A (en) | A kind of detection reagent, kit and method detecting anti-liver cancer and anti-natural antibody | |
| CN114113594B (en) | Antigen diluent and immunodeficiency virus detection kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |